The longterm objectives of this research are to explore and define the roles of reoxygenation and recruitment/redistribution in the response of primarily neoplastic cells to ionizing radiation. Specific aims are to investigate the kinetics of reoxygenation during fractionated irradiation; to investigate reoxygenation during and after treatment of tumors with cytotoxic drugs; to determine whether the kinetics of reoxygenation are affected by radiation combined with drugs; to determine whether cell migration might be detected especially in heterotypic systems by immunofluorescent identification techniques; to determine the effect of radiation on net inward migration of cells into spheroids; to study by different methods migration of cells outward from the centers of spheroids and how this is affected by irradiation; to study the effects of inhibitors of cell motility on tumor cell migration; to complete the development of SACCAS (the Stanford Automated Cell/Colony Autoradiographic Scanner); to investigate the differential radiosensitivity of P and Q cell; to determine whether Q cells are recruited after irradiation, after chemotherapy, and after combinations of the two; to develop algorithms for the analysis of multifield time-lapse movies in order to extract data on cell generation times, colony abortion probabilities, and related statistics; and to correlate 2-dimensional with 3-dimensional migration of EMT6 and/or RIF-1 tumor cells. The methodology includes the use of the paired survival curve method for studying reoxygenation kinetics, the use of multicellular tumor cell spheroids in either homotypic or heterotypic combinations with indicator cells as an in vitro model in order to study migration and thereby assess its role in tumor reoxygenation (detecting the indicator cells either autoradiographically or by immunofluorescence), differentiating between P and Q cells by the ability of the former to incorporate 3HTdR and identifying P cells and their descendants autoradiographically using SACCAS, and producing multifield time-lapse movies of colony growth in vitro by a novel automated technique.